Viral gene delivery, as a molecular biology tool or as a potential approach to gene-based therapies, is without doubt the most efficient method of DNA delivery, or transfection, found to date. Although viral vectors are generally more efficient than non-viral vectors, several disadvantages of viral vectors (e.g. antigenicity, production cost, limited size of cargo, etc) mean that non-viral chemical based delivery systems represent a very attractive alternative, especially because of their relatively low cost and procedural simplicity. Despite numerous improvements, however, the in vivo efficacy of non-viral vectors still needs to be increased for both clinical and research purposes, which have been most widely studied to date.
Among the many non-viral vectors (for a review see M A Mintzer and E E Simanek (Chem. Rev., 2009, 109, 259-302)), cationic lipids are perhaps the class of compounds that have been most widely studied to date. For detailed reviews see B Martin et al., (Curr. Pharm. Design, 2005, 11, 375-394) and S Bhattacharya and A Bajaj (Chem. Commun., 2009, 4632-4656).
As is well-known and well-understood in the art cationic lipids typically comprise three main parts: a lipophilic component attached through a linking moiety to a positively charged, polar head group. The positively charged, polar head group is typically the result of protonation of one or more amino groups, or may arise by the provision of quaternary amines, which bears a permanent positive charge.
When cationic lipids are mixed with DNA or RNA, or other molecules, in an aqueous solution, electrostatic and hydrophobic interactions are known to lead to organization via a multi-step mechanism into a liposome-like complex known as a lipoplex (see B Martin et al., infra). At the end of this multi-step process, the DNA or RNA is condensed, generally the cationic lipids totally envelop the plasmid (providing shielding from nucleases in the surrounding environment) (see B Martin et al., infra). Use of an excess of cationic lipid provides an overall a positive charge which is postulated to mediate cellular uptake (via non-specific endocytosis) following an interaction with negatively charged cell surface structures such as phospholipids, or heparin sulphates or other proteoglycans.
The first reported cationic lipid used for DNA delivery DOTMA, (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl-ammonium chloride), was reported in 1987. Now, an enormous range of cationic lipids have been synthesized (see B Martin et al. and S Bhattacharya and A Bajaj, infra) and several are commercially available. These include Lipofectamine™ 2000 (Invitrogen) and Effectene® Transfection Reagent (Qiagen).
Lipoplex formulation is often assisted by the addition of a neutral surfactant, such as dioleyl phosphatidyl ethanolamine (DOPE), which is believed to improve the transfection abilities of the mixture (H Farhood et al., Biochim. Biophys. Acta, 1995, 1235, 289-295). Due to its fusogenic properties (H Farhood et al., (infra); H Ellens, et al. (Biochemistry, 1986, 25, 4141-7); and I Koltover. et al. (Science, 1998, 281, 78-81)) this so-called “co-lipid”, or helper lipid, appears to drive lipoplex assembly (possibly promoting a transition from lamellar to hexagonal phase) by increasing the release of counterions, although DOPE itself is not required for lipoplex assembly. The presence of DOPE is thought to loosen the binding of the cationic lipid to the DNA and enhance endosomal escape of the lipoplexes in to the cytoplasm, a step which is probably the most important in the entire transfection process.
Typically, the head groups found in cationic lipids are nitrogen-based motifs which are protonated (quaternary amines) or will become protonated at physiological pH. The resultant positive charge assists in the binding and packing of the phosphate diester backbone of DNA and RNA. For example, M Furuhata et al. (Int. J. Pharm., 2006, 316, 109-116) describe the design, synthesis and evaluation of in vitro gene delivery efficacy of a series of cationic lipids constituted by using 3,5-bis(dodecyloxy)benzamide as the lipid component, the amide group of which is connected to the C-termini of four different oligo-arginines through poly(ethylene glycol) (PEG) spacers.
Generally the lipophilic component is composed of two long chain fatty acids or a cholesterol-based derivative. Other than with cholesterol-based cationic lipids, the hydrophobic moiety of cationic lipids generally contains unsaturated or saturated alkyl or acyl (alkylcarbonyl) chains, with a chain length of typically about 12-18 carbon atoms. Long saturated tails tend to display relatively strong intermolecular interactions and a low propensity for mixing with neutral helper lipids such as DOPE. The addition of double bonds leads to less compact packing. Hence, hydrocarbon tail length and saturation affect lipoplex intra-dynamics and ultimately the packing efficiency of DNA. Although the majority of cationic lipids have two chains, on-going research has looked at the use of single chain detergents that are capable of dimerisation via oxidation and tripod-like cationic lipids (A Unciti-Broceta et al., J. Med. Chem., 2008, 51, 4076-4084). Cholesterol-based tails are often used as an alternative to aliphatic chains since cholesterol is rigid and biodegradable and can be considered to be a structural mimic of two long fatty acid chains. Cholesterol is also used as an alternative co-lipid to DOPE.
An acknowledged class of cationic lipids is constituted by compounds known as gemini surfactants (for a mini review see A J Kirby et al., Angew. Chem. Int. Ed., 2003, 42, 1448-1457). Unlike most other cationic lipids, gemini surfactants contain two head groups and two aliphatic chains, which are linked by a rigid or flexible spacer. The presence of two head groups (each with an associated aliphatic tail) is described by A J Kirby et al. as having greatly enhanced surfactant properties relative to the corresponding “monovalent” (i.e. single chain, single head group) compounds.
As the packing properties of cationic lipids are important for optimal condensation of DNA (see V A Bloomfield, Curr. Opin. Struct. Biol., 1996, 6, 334-341) the structures of cationic lipids are generally very carefully designed to enable effective DNA-binding and lipoplex formation. Indeed the prior art is replete with reports of studies into the rational design of cationic lipids by way of modification of the three main parts (i.e. the lipophilic component, the linking motif and the head group), which studies have allowed the elucidation of structure-activity relationships. For example it is postulated that the larger the imbalance between the cross-sectional area of the cationic (small) end and the large hydrophobic moiety, the more ‘cone-shaped’ the cationic lipid, which is believed by some to create greater instability in the resulting lipid assembly. Such instability can lead to improved transfection with DNA or RNA release in to the cytoplasm improved.
Hydrophobic and hydrophilic portions of cationic lipids have generally been joined using amide, ether, ester or carbamate bonds, although there is no optimal bond. Ether-containing cationic lipids are more stable than ester-containing counterparts with carbamate-containing lipids viewed as a good balance between stability and toxicity. The linking bond may be considered to control the cationic amphiphilic lipid's stability, thereby controlling the balance between persistence and toxicity. In terms of linker design there are a great number of reports including the use of photosensitive linkers and the incorporation of environmental sensitive groups, where intracellular hydrolysis leads to controlled DNA delivery at defined stages during intracellular lipoplex trafficking.
Notwithstanding all the prior art, however, there is still a requirement for alternative cationic lipids that offer one or more properties such as reduced cell toxicity, promotion of endosomal escape of molecules, e.g. nucleic acids, and, where the molecule to be transfected is a DNA molecule, folding of that DNA.
Despite the vast amount of work undertaken to date in the field of cationic lipids, therefore, it is desired to develop further cationic lipids capable of ameliorating or obviating one or more of the problems of in vivo efficacy of the transfection process, toxicity, cost and simplicity of design.